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β-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts
Theriogenology 79 (2013) 284–290
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Possible involvement of Wnt/b-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts Kyung Tae Lim a, Mukesh Kumar Gupta a, b, *, Sung Ho Lee a, Yoon Hee Jung a, Dong Wook Han a, Hoon Taek Lee a, * a b
Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul, Korea Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2012 Received in revised form 21 August 2012 Accepted 27 August 2012
The Wnt/b-catenin signaling pathway plays essential roles in the regulation of cell fate and polarity during embryonic development of many animal species. This study investigated the possible involvement of Wnt/b-catenin signaling pathway during hatching and trophectoderm (TE) development in pig blastocysts. Results showed that b-catenin and DVL3, the key mediators of Wnt/b-catenin signaling, disappeared from the nucleus after blastocyst hatching. Specific inhibition of Wnt/b-catenin signaling pathway, by Dickkopf-1, increased the rate of blastocyst hatching, total nuclear number per blastocyst, and reduced the ratio of inner cell mass (ICM):TE (P < 0.05). In contrast, specific activation of the Wnt/b-catenin signaling pathway, by lithium chloride, reduced the rate of blastocyst hatching, total nuclear number per blastocyst, and increased the ratio of ICM:TE (P < 0.05). The change in the ICM:TE ratio was associated with the change in the number of TE cells but not the ICM cells. Activation or inhibition of Wnt/b-catenin signaling and b-catenin nuclear accumulation, by lithium chloride or Dickkopf-1, also altered the expression of CDX2. These data therefore, suggest the possible involvement of Wnt/b-catenin signaling in regulating hatching and TE fate during the development of pig blastocyst. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Blastocyst hatching Trophectoderm Dickkopf-1 DVL3 b-catenin CDX2
1. Introduction In mammals, the earliest lineage differentiation occurs at the blastocyst stage with the establishment of morphologically distinguishable inner cell mass (ICM) and trophectoderm (TE). Two models, the inside-outside model and the polarity model, have been proposed for the ICM and TE lineage differentiation in the blastocyst (see [1] for detailed review). However, involvement of different cell signaling pathways in early determination of cell fate
* Corresponding author. Tel.: þ91 661 2462294; fax: þ91 661 2462281. * Alternate corresponding author. Tel.: þ82 2 4503675; fax: þ82 2 4578488. E-mail addresses: [email protected] (M.K. Gupta), htl3675@konkuk. ac.kr (H.T. Lee). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.08.018
remains elusive. Recent studies show that Hippo signaling pathway can regulate the spatial expression of TEA domain family transcription factor, Tead4, to regulate the expression of Caudal type homeobox 2 (Cdx2) transcriptional factor and thereby, the TE fates of the blastomeres [2]. The components of Hippo signaling pathway can also interact with the Wnt/ b-catenin signaling pathway [3]. The Wnt/b-catenin signaling pathway has been implicated in a wide range of biological processes including cell proliferation, differentiation, epithelial-mesenchymal communication, specification of cell fate, induction of the body axis, and determination of embryonic patterning in both invertebrates and vertebrates [4–6]. This pathway has been extensively studied in Drosophila, Caenorhabditis, Zebrafish, Xenopus, and mouse, and was demonstrated to be important for morphogenesis, anterior-posterior axis development and neural tube patterning [6–8]. In classic
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canonical pathway, binding of Wnt proteins to their Frizzled family of cell surface receptors results in the liberation of b-catenin, which enters the nucleus to activate transcription of Wnt target genes. Targeted deletion of specific Wnt genes in mouse lead to specific developmental defects such as placental defect in Wnt2-deleted [9] and Wnt7b-deleted mouse [10] and female infertility, abnormal uterine patterning and defects of limb polarity in Wnt 7a-deleted mouse [11,12]. Wnt was also expressed in uterine epithelium of female mouse [10–12]. It was therefore, suggested that Wnt/b-catenin signaling pathway might be an intimate cross-talk mechanism between the blastocyst and uterus to coordinate the molecular events in blastocyst and the receptivity events in uterus during the peri-implantation period (see [13] for detailed review). The transcripts and proteins of Wnt ligands, receptors, and several other intracellular components of the Wnt/bcatenin signaling pathway have also been found in oocytes and embryos of mouse and Rhesus monkey [14–20]. However, there is no concrete evidence for their functional activity in preimplantation stage embryos although the function of several Wnt molecules is well recognized during postimplantation embryonic development [6,7,21,22]. Several studies have shown that mouse embryos lacking the b-catenin or expressing the truncated b-catenin, the key component of the Wnt/b-catenin signaling pathway, undergo normal maturation, fertilization, and early two-cell stage development [21]. Interestingly, however, such embryos exhibited premature epithelial-mesenchymal transition [22] and early gastrulation patterning defects [23,24]. Furthermore, b-catenin was found to be expressed in the nuclei of ICM cells but not in the TE cells [19,25]. The activation of Wnt signaling pathway also promoted the proliferation and maintenance of pluripotency in ICM-derived embryonic stem cells [4,25,26]. A previous study in mouse has also reported that b-catenin was localized in the nucleus of the outer cells in morula and in the TE cells of unhatched blastocyst but disappeared in the hatched blastocyst until the postimplantation development [27]. It is, therefore, likely that Wnt/b-catenin signaling might be involved in the hatching and/or TE differentiation during preimplantation embryonic development [27]. Thus, using specific chemical inhibitor and activator, the present study explored the possible involvement of Wnt/bcatenin signaling pathway in regulating the hatching and TE differentiation in pig blastocysts. Pig blastocyst was chosen for the study because limited information is available for this species despite their well recognized application in biomedicine and xenotransplantation. Specific chemical inhibitor and activator of Wnt/b-catenin signaling pathway were used to simulate loss-of-function and gainof-function study.
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abattoir-derived pig ovaries were randomly distributed to different groups. 2.1. In vitro production and culture (IVC) of pig blastocysts Pig blastocysts were produced by in vitro fertilization of abattoir ovary-derived oocytes essentially as we described earlier [28]. Briefly, cumulus oocyte complexes, aspirated from prepubertal pig ovaries, were matured in vitro in groups of 50 in 500 mL of Tissue Culture Medium 199 with Earle’s salts (TCM-199; Gibco BRL, Grand Island, NY, USA) supplemented with 25 mM NaHCO3, 10% (vol/vol) porcine follicular fluid, 0.57 mM cysteine, 0.22 mL/mL sodium pyruvate, 25 mg/mL gentamicin sulfate, 0.5 mL/mL FSH (Folltropin V; Vetrepharm, Ontario, Canada), 1 mg/mL estradiol-17b, and 10 ng/mL epidermal growth factor under mineral oil at 38.5 C in a humidified atmosphere of 5% CO2 in air for 42 h. Matured oocytes were subsequently denuded of cumulus cells using 0.1% (wt/vol) hyaluronidase and placed into groups of approximately 10 to 15 oocytes per 50 mL droplets of the fertilization medium (modified Tris buffered medium containing 1 mM caffeine sodium benzoate and 0.1% BSA). Boar sperm, retrieved from cauda epididymis of abattoir-derived boar testes, were subjected to swim-up in Sp-TALP medium for 10 min and were added to the fertilization droplet to obtain a final sperm concentration of 5 105 cells per mL. In each experimental replicate, the semen was derived from the same testis for all the experimental groups. Sperm and oocytes were coincubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 6 h. At the end of the coincubation period, presumptive zygotes were cultured in North Carolina State University 23 medium supplemented with 0.4% BSA for 5 to 8 days. Hatching ability of embryos was expressed as: number of embryos hatched out of zona pellucida/total number of embryos cultured 100. 2.2. Differential staining of ICM and TE Differential staining of cells within ICM and TE was performed essentially as we described earlier [29]. Briefly, blastocysts were removed of their zona pellucida by treatment with 0.5% (wt/vol) pronase and were subsequently exposed to a 1:5 dilution of rabbit anti-pig whole serum for 1 h. Blastocysts were then washed three times for 5 min and exposed to 1:10 dilution of guinea pig complement containing 10 mg/mL propidium iodide and 10 mg/mL bisbenzimide for 1 h. After brief washing, the stained embryos were mounted on clean glass slides and examined under ultraviolet light using an epifluorescent microscope. Blue and red cells were counted as cells from the ICM and TE, respectively. Percentage of cells in ICM or TE was calculated as: number of red cells or blue cells/total number of blue and red cells 100.
2. Materials and methods
2.3. Immunocytochemistry
All chemicals were purchased from Sigma-Aldrich Co., (St. Louis, MO, USA) unless otherwise specifically indicated. Each experiment consisted of at least four replicates and in each replication, oocytes from the same collection of
Analyses of blastocysts for the expression and localization of DVL3, b-catenin, and CDX2 proteins were performed by immunocytochemistry as we described earlier [30] with modifications. Briefly, blastocysts were rinsed in
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pre-extraction solution (130 mM KCl, 25 mM HEPES, 3 mM MgCl2, and 0.06% Triton X-100) and were fixed and permeabilized in PBS containing 4% (wt/vol) paraformaldehyde and 0.5% (vol/vol) Triton X-100. Blastocysts were then incubated overnight at 4 C in blocking solution (2% BSA, 2% skim milk, 0.2% Tween-20) to block nonspecific binding of antibodies and were subsequently incubated with the primary antibodies against DVL3 (1:50; Cell Signaling Technology, Inc., Danvers, MA, USA), b-catenin (1:100; Abcam, Cambridge, MA, USA), or CDX2 (1:200; Millipore, Billerica, MA, USA), which were raised in rabbit host using synthetic peptide epitopes specific to the respective proteins. Bound primary antibodies were localized with FITC-conjugated anti-rabbit IgG second antibody (1:250; Molecular Probes, Leiden, The Netherlands). Blastocysts were counter stained with nuclear fluorochrome, propidium iodide (200 mg/mL) for 15 min, mounted on glass slides using antifade mounting medium (FluoroGuard; Biorad, Hercules, CA, USA) and observed under 633 nm with a confocal laser scanning microscope (Fluoview FV1000-ASWv1.5; Tokyo, Japan). Experiments were repeated four times with approximately 5 to 8 embryos in each replication. Second antibody control (negative control) was included in each replication wherein immunocytochemistry was performed essentially as described above except that the primary antibody was omitted.
2.4. Experimental design 2.4.1. Experiment 1: Expression of DVL3 and b-catenin during expansion and hatching of pig blastocysts In the first set of experiments, we investigated the expression and localization of DVL3 and b-catenin proteins which are critical mediators of the Wnt/b-catenin signaling pathway [31–33]. Nonhatched expanded blastocysts and hatched blastocysts were collected on Day 7 of IVC and were analyzed for the expression of DVL3 and b-catenin by immunocytochemistry. 2.4.2. Experiment 2: Effect of activating or inhibiting Wnt/ b-catenin signaling pathway on hatching ability of pig blastocysts In this set of experiments, to investigate the importance of Wnt/b-catenin signaling pathway during hatching of pig blastocysts, Day 6 expanded blastocysts were cultured in the IVC medium supplemented with and without Dickkopf-1 (Dkk1; 10 mg/mL; R&D Systems, Minneapolis, MN, USA) or lithium chloride (LiCl; 10 mM) which are a well known specific inhibitor (Dkk1) and activator (LiCl) of the Wnt/b-catenin signaling pathway, respectively [27,34,35]. LiCl mimics Wnt by inhibiting the serine kinase activity of GSK3 [27] whereas Dkk1 binds to the LRP coreceptor and thereby, blocks the activation of the Wnt
Fig. 1. Localization of DVL3 and b-catenin (inset) proteins in expanded and hatched blastocysts. Panel 1: Green color shows the localization of bound primary antibodies captured with FITC-conjugated secondary antibody. Panel 2: Red color shows the propidium iodide (PI)-stained nuclei. Panel 3: Merged panels 1 and 2. In the negative control group, expanded/hatched blastocysts were stained with secondary antibody (primary antibody omitted) similar to other experimental groups. Magnification: DVL3 400; b-catenin 600.
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3. Results and discussion
possible involvement of Wnt/b-catenin signaling pathway during the hatching and/or early epithelial differentiation of pig blastocyst. To validate the importance of Wnt/b-catenin signaling pathway during hatching and/or early epithelial differentiation of pig blastocyst, we either activated or inhibited the Wnt signaling using a specific activator (LiCl) and inhibitor (Dkk1). We observed that culture of Day 6 blastocysts in the presence of Dkk1 inhibited the Wnt/b-catenin signaling pathway, as revealed by reduced accumulation of b-catenin (Supplementary Fig. 1). The Dkk1-treated blastocysts showed increased (P < 0.05) ability to hatch on Day 7 (57.5 1.9 vs. 28.0 2.7%) and Day 8 (83.6 3.6 vs. 58.7 2.5%) of IVC (Fig. 2). On the contrary, culture of Day 6 blastocysts in the presence of LiCl increased the accumulation of b-catenin (Supplementary Fig. 1) but reduced the ability of blastocysts to hatch on Day 7 (3.1 1.1%) and Day 8 (15.2 1.4%) of IVC (Fig. 2). Thus, hatching ability of pig blastocyst was inversely corelated with the activation of Wnt/b-catenin signaling pathway. A previous study in mouse suggested that inhibition of Wnt/b-catenin during implantation might provide signal to blastocyst to begin hatching and to synchronize the preimplantation embryonic development with the differentiation of the uterus [27]. Next, we evaluated if the altered hatching ability of Dkk1- and LiCl-treated blastocysts were associated with altered development and differentiation of TE cells because of inhibition or activation of the Wnt/b-catenin signaling pathway. Because earliest epithelial differentiation of pig embryos are observed on Day 5 of IVC [36], we treated them with Dkk1 or LiCl for 24 h and then transferred to fresh media for further culture until Day 7. Results showed that Dkk1-treated blastocysts not only had increased (P < 0.05) hatching ability but also contained increased (P < 0.05) number of blastomeres compared with nontreated controls (Fig. 3). Furthermore, Dkk1-treated blastocysts displayed significantly lower ICM:TE ratio than
To study the role of the Wnt/b-catenin signaling pathway during hatching and early epithelial differentiation of pig blastocysts, we first examined the expression and distribution of b-catenin and DVL3 in the blastocyst during expansion and hatching. The b-catenin is a central component in the Wnt/b-catenin signaling pathway whereas DVL is a key transducer of divergent Wnt pathways and releases b-catenin from degradation during Wnt/ b-catenin signaling [31–33]. Previous studies have shown that, in an unstimulated state, b-catenin and DVL3 localizes as punctate vesicular structures in the cytoplasm of cells but translocates to the nucleus upon stimulation by Wnt signals [31,32,37–39]. We observed that, similar to those reported for mouse embryos [27], both DVL3 and b-catenin were clearly expressed in the nuclei of expanded blastocysts but disappeared in hatched blastocysts (Fig. 1). Thus, the Wnt/b-catenin signaling pathway was active in expanded blastocyst but was inhibited in hatched blastocysts. We also observed that b-catenin was also localized at the cell-cell borders of both expanded and hatched blastocysts. In tissue culture cells, cadherin had a potential role in sequestering signaling b-catenin at the plasma membrane [27,40]. These data therefore, suggest the
Fig. 2. Activation or inhibition of the Wnt/b-catenin pathway influences the hatching ability of pig blastocysts. Day 6 blastocysts were cultured in the absence (control; closed box) or presence of specific inhibitor (Dickkopf-1; open box) or activator (lithium chloride; shaded box) of Wnt/b-catenin signaling pathway and were evaluated for the percentage of hatched blastocysts after 24 h (Day 7) or 4 h (Day 8) of in vitro culture. Bars with different letters (a, b, c) differ significantly (P < 0.05) among respective groups.
signaling pathway by Wnt signals [34,35]. Blastocysts were evaluated for their hatching ability on Day 7 and 8 of IVC. The concentrations of the inhibitor/activator, used in the study, were based our preliminary experiments (Supplementary Table 1). The ability of Dkk1 and LiCl to modulate the Wnt/b-catenin signaling pathway was confirmed by evaluating the expression of b-catenin in the Dkk1/LiCl-treated blastocysts by immunocytochemistry. 2.4.3. Experiment 3: Effect of activating or inhibiting Wnt/ b-catenin signaling pathway on early epithelial differentiation in pig blastocysts In this set of experiments, to investigate the role of Wnt/ b-catenin signaling pathway on early epithelial differentiation of pig blastocysts, Day 5 embryos (stage at which early epithelial differentiation begins in pig embryos [36]) were cultured in the IVC medium supplemented with and without Dkk1 (10 mg/mL) or LiCl (10 mM) for 24 h (Day 6) and were subsequently cultured in the absence of Dkk1 and LiCl for the next 24 h (Day 7). Blastocysts were evaluated for their hatching ability, total cell number per blastocyst, ICM, TE, and the expression of CDX2. 2.5. Statistical analyses Statistical analyses were carried out using SAS software Version 8.2 (Statistical Analysis System Inc., Cary, NC, USA). Embryo development data was analyzed by chi-square test or ANOVA and the means of cell number were compared by ANOVA followed by Bonferroni multiple pair wise comparison after testing for normality (Kolmogorov– Smirnov test with Lillie for correction). Arc-sine transformation was performed before analyzing the percentage data. Data are presented as mean SEM. Differences at P < 0.05 were considered significant.
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Fig. 3. Activation or inhibition of the Wnt/b-catenin pathway influences the early epithelial differentiation in pig embryos. Day 5 embryos were cultured in the absence (control; closed box) or presence of specific inhibitor (Dickkopf-1 [Dkk1]; open box) or activator (lithium chloride [LiCl]; shaded box) of Wnt/b-catenin signaling pathway for 24 h and were subsequently cultured for another 24 h in the activator/inhibitor-free medium. (A) Time course of blastocyst expansion and hatching. (B) Hatching ability and total cell number/blastocyst. Bars with different letters (a, b, c) differ significantly (P < 0.05) among the respective groups. (C) Inner cell mass (ICM; blue color) and trophectoderm (TE; pink color) cells in blastocysts. (D) Expression and localization of CDX2 protein in pig blastocysts, as revealed by immunocytochemistry using specific antibody against CDX2. Green color shows the localization of bound primary antibodies captured with FITCconjugated secondary antibody. Red color shows the propidium iodide-stained nuclei. Magnification 400.
those of nontreated controls (Table 1; Fig. 3). In other words, Dkk1-treated blastocysts had a significantly higher proportion of TE cells to ICM cells compared with that of other groups (Table 1). In sharp contrast, LiCl-treated blastocysts showed significantly reduced hatching ability, contained reduced number of blastomeres, and had the lower proportion of TE cells to ICM cells than those of other groups (Fig. 3; Table 1). Of note, the change in proportion of ICM and TE cells in Wnt-inhibited blastocysts were related to increase in total cell number and increase in TE cells and the total number of ICM cells remained similar. Conversely, the change in proportion of ICM and TE cells in Wntactivated blastocysts were related to reduced total cell number and reduced TE cells. Thus, inhibition of Wnt/bcatenin signaling pathway probably increased the propensity of blastomeres toward the fate of TE and thereby, increased the total number of cells in blastocyst.
These results are in line with previous reports that activation of the Wnt signaling pathway was sufficient to maintain the pluripotency and proliferation of ICM-derived embryonic stem cells [4,25,26]. Differentiation and proliferation of TE cells is regulated by the transcription factor Cdx2 whose expression in turn is regulated by the transcription factor Tead4 [41]. Recent studies show that Hippo signaling pathway can regulate the spatial expression of Tead4 to regulate the expression of Cdx2 and TE fates of the blastomeres [2]. The Hippo signaling pathway can also interact with the Wnt/b-catenin signaling via the regulation of DVL activity and nuclear accumulation of b-catenin [3]. We found that modulation of Wnt/b-catenin signaling and b-catenin nuclear accumulation, by LiCl or Dkk1, not only affected the TE proliferation but also altered the expression of CDX2. The nuclei of LiCl-treated blastocysts showed reduced and irregular
Table 1 Effect of Dkk1 and LiCl on ICM and TE. Groups
Control Dkk1 LiCl
Cells, N (mean SEM)
Cells, % (mean SEM)
ICM:TE ratio
ICM
TE
ICM
TE
14.7 1.6a 13.7 0.8a 13.4 0.8a
65.1 3.7b 86.1 9.1a 48.0 3.2c
18.0 1.0b 14.5 1.0c 22.9 1.0a
82.0 1.0b 85.1 1.0a 78.7 1.5c
Values with different superscripts within each column differ significantly (P < 0.05). Abbreviations: Dkk1, Dickkopf-1; ICM, inner cell mass; LiCl, lithium chloride; TE, trophectoderm.
0.22 0.1a 0.17 0.1b 0.29 0.1c
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expression of CDX2 compared with those of control and Dkk1-treated blastocysts (Fig. 3). These data therefore, reinforce the possible involvement of Wnt/b-catenin signaling in regulating TE fate during the development of pig blastocyst. Future study should determine if the Wnt/bcatenin signaling pathway directly interacted with the Hippo signaling pathway in embryos to regulate the expression of Cdx2 and hence, the TE fate of the blastomeres. It might also be noted that, in our study, we used in vitro-produced embryos, which are known to have lower development potential than their in vivo counterparts. It remains to be determined if the Wnt/b-catenin signaling pathway behaved similarly in the in vivo-produced embryos. In conclusion, our study suggests that Wnt/b-catenin signaling might possibly be involved in the hatching and TE differentiation of pig blastocyst, providing the participation of Wnt/b-catenin signaling in preimplantation development. The results of this study will not only have implications in modulating the ICM:TE ratio of in vitro produced animal embryos (such as cloned embryos) but also will be of use in infertility treatment via the promotion of hatching in difficult-to-hatch blastocysts. Further studies should unravel the possible cross talk of Wnt/b-catenin signaling to other signaling pathways in hatching and TE fate determination. Acknowledgments This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (#PJ0090142012)” and BioGreen 21 Program (#PJ0080962012), Rural Development Administration, Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. theriogenology.2012.08.018. References [1] Zernicka-Goetz M, Morris SA, Bruce AW. Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nat Rev Genet 2009;10:467–77. [2] Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, et al. The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 2009;16:398–410. [3] Varelas X, Miller BW, Sopko R, Song S, Gregorieff A, Fellouse FA, et al. The Hippo pathway regulates Wnt/beta-catenin signaling. Dev Cell 2010;18:579–91. [4] Sokol SY. Maintaining embryonic stem cell pluripotency with Wnt signaling. Development 2011;138:4341–50. [5] Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP. Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Dev Growth Differ 2011;53:843–56. [6] van Amerongen R, Nusse R. Towards an integrated view of Wnt signaling in development. Development 2009;136:3205–14. [7] Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR, Bradley A. Requirement for Wnt3 in vertebrate axis formation. Nat Genet 1999;22:361–5. [8] Petersen CP, Reddien PW. Wnt signaling and the polarity of the primary body axis. Cell 2009;139:1056–68.
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Supplementary Fig. 1. Expression of b-catenin in expanded blastocysts cultured in the absence (control; A) or presence of specific inhibitor (Dickkopf-1 [Dkk1]; B) or activator (lithium chloride [LiCl]; C) of the Wnt/b-catenin signaling pathway. Green color shows the localization of bound primary antibodies captured with FITC-conjugated secondary antibody. Red color shows the propidium iodide [PI]-stained nuclei. In the negative control group (D), blastocysts were stained with secondary antibody (primary antibody omitted) similar to other experimental groups. Magnification 600.
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Supplementary Table 1 Dose-dependent effect of LiCl on the hatching ability of Day 6 pig blastocysts. Concentration (mM)
Blastocysts, N
Hatching ability (mean SEM)
Control 0.01 0.1 1.0 10
32 33 32 34 32
56.3 36.4 28.1 11.8 3.1
2.5 2.7 1.7 3.6 1.3
(18) (12) (9) (4) (1)
Day 6 blastocysts were cultured in the absence (control) or presence of LiCl and evaluated for the percentage of hatched blastocysts after 24 h (Day 7). The experiment was repeated four times. Values within parenthesis indicate the number of embryos. Hatching ability for all concentrations differed significantly (P < 0.05). Abbreviation: LiCl, lithium chloride.
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Possible involvement of Wnt/b-catenin signaling pathway in hatching and trophectoderm differentiation of pig blastocysts Kyung Tae Lim a, Mukesh Kumar Gupta a, b, *, Sung Ho Lee a, Yoon Hee Jung a, Dong Wook Han a, Hoon Taek Lee a, * a b
Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul, Korea Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2012 Received in revised form 21 August 2012 Accepted 27 August 2012
The Wnt/b-catenin signaling pathway plays essential roles in the regulation of cell fate and polarity during embryonic development of many animal species. This study investigated the possible involvement of Wnt/b-catenin signaling pathway during hatching and trophectoderm (TE) development in pig blastocysts. Results showed that b-catenin and DVL3, the key mediators of Wnt/b-catenin signaling, disappeared from the nucleus after blastocyst hatching. Specific inhibition of Wnt/b-catenin signaling pathway, by Dickkopf-1, increased the rate of blastocyst hatching, total nuclear number per blastocyst, and reduced the ratio of inner cell mass (ICM):TE (P < 0.05). In contrast, specific activation of the Wnt/b-catenin signaling pathway, by lithium chloride, reduced the rate of blastocyst hatching, total nuclear number per blastocyst, and increased the ratio of ICM:TE (P < 0.05). The change in the ICM:TE ratio was associated with the change in the number of TE cells but not the ICM cells. Activation or inhibition of Wnt/b-catenin signaling and b-catenin nuclear accumulation, by lithium chloride or Dickkopf-1, also altered the expression of CDX2. These data therefore, suggest the possible involvement of Wnt/b-catenin signaling in regulating hatching and TE fate during the development of pig blastocyst. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Blastocyst hatching Trophectoderm Dickkopf-1 DVL3 b-catenin CDX2
1. Introduction In mammals, the earliest lineage differentiation occurs at the blastocyst stage with the establishment of morphologically distinguishable inner cell mass (ICM) and trophectoderm (TE). Two models, the inside-outside model and the polarity model, have been proposed for the ICM and TE lineage differentiation in the blastocyst (see [1] for detailed review). However, involvement of different cell signaling pathways in early determination of cell fate
* Corresponding author. Tel.: þ91 661 2462294; fax: þ91 661 2462281. * Alternate corresponding author. Tel.: þ82 2 4503675; fax: þ82 2 4578488. E-mail addresses: [email protected] (M.K. Gupta), htl3675@konkuk. ac.kr (H.T. Lee). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.08.018
remains elusive. Recent studies show that Hippo signaling pathway can regulate the spatial expression of TEA domain family transcription factor, Tead4, to regulate the expression of Caudal type homeobox 2 (Cdx2) transcriptional factor and thereby, the TE fates of the blastomeres [2]. The components of Hippo signaling pathway can also interact with the Wnt/ b-catenin signaling pathway [3]. The Wnt/b-catenin signaling pathway has been implicated in a wide range of biological processes including cell proliferation, differentiation, epithelial-mesenchymal communication, specification of cell fate, induction of the body axis, and determination of embryonic patterning in both invertebrates and vertebrates [4–6]. This pathway has been extensively studied in Drosophila, Caenorhabditis, Zebrafish, Xenopus, and mouse, and was demonstrated to be important for morphogenesis, anterior-posterior axis development and neural tube patterning [6–8]. In classic
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canonical pathway, binding of Wnt proteins to their Frizzled family of cell surface receptors results in the liberation of b-catenin, which enters the nucleus to activate transcription of Wnt target genes. Targeted deletion of specific Wnt genes in mouse lead to specific developmental defects such as placental defect in Wnt2-deleted [9] and Wnt7b-deleted mouse [10] and female infertility, abnormal uterine patterning and defects of limb polarity in Wnt 7a-deleted mouse [11,12]. Wnt was also expressed in uterine epithelium of female mouse [10–12]. It was therefore, suggested that Wnt/b-catenin signaling pathway might be an intimate cross-talk mechanism between the blastocyst and uterus to coordinate the molecular events in blastocyst and the receptivity events in uterus during the peri-implantation period (see [13] for detailed review). The transcripts and proteins of Wnt ligands, receptors, and several other intracellular components of the Wnt/bcatenin signaling pathway have also been found in oocytes and embryos of mouse and Rhesus monkey [14–20]. However, there is no concrete evidence for their functional activity in preimplantation stage embryos although the function of several Wnt molecules is well recognized during postimplantation embryonic development [6,7,21,22]. Several studies have shown that mouse embryos lacking the b-catenin or expressing the truncated b-catenin, the key component of the Wnt/b-catenin signaling pathway, undergo normal maturation, fertilization, and early two-cell stage development [21]. Interestingly, however, such embryos exhibited premature epithelial-mesenchymal transition [22] and early gastrulation patterning defects [23,24]. Furthermore, b-catenin was found to be expressed in the nuclei of ICM cells but not in the TE cells [19,25]. The activation of Wnt signaling pathway also promoted the proliferation and maintenance of pluripotency in ICM-derived embryonic stem cells [4,25,26]. A previous study in mouse has also reported that b-catenin was localized in the nucleus of the outer cells in morula and in the TE cells of unhatched blastocyst but disappeared in the hatched blastocyst until the postimplantation development [27]. It is, therefore, likely that Wnt/b-catenin signaling might be involved in the hatching and/or TE differentiation during preimplantation embryonic development [27]. Thus, using specific chemical inhibitor and activator, the present study explored the possible involvement of Wnt/bcatenin signaling pathway in regulating the hatching and TE differentiation in pig blastocysts. Pig blastocyst was chosen for the study because limited information is available for this species despite their well recognized application in biomedicine and xenotransplantation. Specific chemical inhibitor and activator of Wnt/b-catenin signaling pathway were used to simulate loss-of-function and gainof-function study.
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abattoir-derived pig ovaries were randomly distributed to different groups. 2.1. In vitro production and culture (IVC) of pig blastocysts Pig blastocysts were produced by in vitro fertilization of abattoir ovary-derived oocytes essentially as we described earlier [28]. Briefly, cumulus oocyte complexes, aspirated from prepubertal pig ovaries, were matured in vitro in groups of 50 in 500 mL of Tissue Culture Medium 199 with Earle’s salts (TCM-199; Gibco BRL, Grand Island, NY, USA) supplemented with 25 mM NaHCO3, 10% (vol/vol) porcine follicular fluid, 0.57 mM cysteine, 0.22 mL/mL sodium pyruvate, 25 mg/mL gentamicin sulfate, 0.5 mL/mL FSH (Folltropin V; Vetrepharm, Ontario, Canada), 1 mg/mL estradiol-17b, and 10 ng/mL epidermal growth factor under mineral oil at 38.5 C in a humidified atmosphere of 5% CO2 in air for 42 h. Matured oocytes were subsequently denuded of cumulus cells using 0.1% (wt/vol) hyaluronidase and placed into groups of approximately 10 to 15 oocytes per 50 mL droplets of the fertilization medium (modified Tris buffered medium containing 1 mM caffeine sodium benzoate and 0.1% BSA). Boar sperm, retrieved from cauda epididymis of abattoir-derived boar testes, were subjected to swim-up in Sp-TALP medium for 10 min and were added to the fertilization droplet to obtain a final sperm concentration of 5 105 cells per mL. In each experimental replicate, the semen was derived from the same testis for all the experimental groups. Sperm and oocytes were coincubated at 38.5 C in a humidified atmosphere of 5% CO2 in air for 6 h. At the end of the coincubation period, presumptive zygotes were cultured in North Carolina State University 23 medium supplemented with 0.4% BSA for 5 to 8 days. Hatching ability of embryos was expressed as: number of embryos hatched out of zona pellucida/total number of embryos cultured 100. 2.2. Differential staining of ICM and TE Differential staining of cells within ICM and TE was performed essentially as we described earlier [29]. Briefly, blastocysts were removed of their zona pellucida by treatment with 0.5% (wt/vol) pronase and were subsequently exposed to a 1:5 dilution of rabbit anti-pig whole serum for 1 h. Blastocysts were then washed three times for 5 min and exposed to 1:10 dilution of guinea pig complement containing 10 mg/mL propidium iodide and 10 mg/mL bisbenzimide for 1 h. After brief washing, the stained embryos were mounted on clean glass slides and examined under ultraviolet light using an epifluorescent microscope. Blue and red cells were counted as cells from the ICM and TE, respectively. Percentage of cells in ICM or TE was calculated as: number of red cells or blue cells/total number of blue and red cells 100.
2. Materials and methods
2.3. Immunocytochemistry
All chemicals were purchased from Sigma-Aldrich Co., (St. Louis, MO, USA) unless otherwise specifically indicated. Each experiment consisted of at least four replicates and in each replication, oocytes from the same collection of
Analyses of blastocysts for the expression and localization of DVL3, b-catenin, and CDX2 proteins were performed by immunocytochemistry as we described earlier [30] with modifications. Briefly, blastocysts were rinsed in
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pre-extraction solution (130 mM KCl, 25 mM HEPES, 3 mM MgCl2, and 0.06% Triton X-100) and were fixed and permeabilized in PBS containing 4% (wt/vol) paraformaldehyde and 0.5% (vol/vol) Triton X-100. Blastocysts were then incubated overnight at 4 C in blocking solution (2% BSA, 2% skim milk, 0.2% Tween-20) to block nonspecific binding of antibodies and were subsequently incubated with the primary antibodies against DVL3 (1:50; Cell Signaling Technology, Inc., Danvers, MA, USA), b-catenin (1:100; Abcam, Cambridge, MA, USA), or CDX2 (1:200; Millipore, Billerica, MA, USA), which were raised in rabbit host using synthetic peptide epitopes specific to the respective proteins. Bound primary antibodies were localized with FITC-conjugated anti-rabbit IgG second antibody (1:250; Molecular Probes, Leiden, The Netherlands). Blastocysts were counter stained with nuclear fluorochrome, propidium iodide (200 mg/mL) for 15 min, mounted on glass slides using antifade mounting medium (FluoroGuard; Biorad, Hercules, CA, USA) and observed under 633 nm with a confocal laser scanning microscope (Fluoview FV1000-ASWv1.5; Tokyo, Japan). Experiments were repeated four times with approximately 5 to 8 embryos in each replication. Second antibody control (negative control) was included in each replication wherein immunocytochemistry was performed essentially as described above except that the primary antibody was omitted.
2.4. Experimental design 2.4.1. Experiment 1: Expression of DVL3 and b-catenin during expansion and hatching of pig blastocysts In the first set of experiments, we investigated the expression and localization of DVL3 and b-catenin proteins which are critical mediators of the Wnt/b-catenin signaling pathway [31–33]. Nonhatched expanded blastocysts and hatched blastocysts were collected on Day 7 of IVC and were analyzed for the expression of DVL3 and b-catenin by immunocytochemistry. 2.4.2. Experiment 2: Effect of activating or inhibiting Wnt/ b-catenin signaling pathway on hatching ability of pig blastocysts In this set of experiments, to investigate the importance of Wnt/b-catenin signaling pathway during hatching of pig blastocysts, Day 6 expanded blastocysts were cultured in the IVC medium supplemented with and without Dickkopf-1 (Dkk1; 10 mg/mL; R&D Systems, Minneapolis, MN, USA) or lithium chloride (LiCl; 10 mM) which are a well known specific inhibitor (Dkk1) and activator (LiCl) of the Wnt/b-catenin signaling pathway, respectively [27,34,35]. LiCl mimics Wnt by inhibiting the serine kinase activity of GSK3 [27] whereas Dkk1 binds to the LRP coreceptor and thereby, blocks the activation of the Wnt
Fig. 1. Localization of DVL3 and b-catenin (inset) proteins in expanded and hatched blastocysts. Panel 1: Green color shows the localization of bound primary antibodies captured with FITC-conjugated secondary antibody. Panel 2: Red color shows the propidium iodide (PI)-stained nuclei. Panel 3: Merged panels 1 and 2. In the negative control group, expanded/hatched blastocysts were stained with secondary antibody (primary antibody omitted) similar to other experimental groups. Magnification: DVL3 400; b-catenin 600.
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3. Results and discussion
possible involvement of Wnt/b-catenin signaling pathway during the hatching and/or early epithelial differentiation of pig blastocyst. To validate the importance of Wnt/b-catenin signaling pathway during hatching and/or early epithelial differentiation of pig blastocyst, we either activated or inhibited the Wnt signaling using a specific activator (LiCl) and inhibitor (Dkk1). We observed that culture of Day 6 blastocysts in the presence of Dkk1 inhibited the Wnt/b-catenin signaling pathway, as revealed by reduced accumulation of b-catenin (Supplementary Fig. 1). The Dkk1-treated blastocysts showed increased (P < 0.05) ability to hatch on Day 7 (57.5 1.9 vs. 28.0 2.7%) and Day 8 (83.6 3.6 vs. 58.7 2.5%) of IVC (Fig. 2). On the contrary, culture of Day 6 blastocysts in the presence of LiCl increased the accumulation of b-catenin (Supplementary Fig. 1) but reduced the ability of blastocysts to hatch on Day 7 (3.1 1.1%) and Day 8 (15.2 1.4%) of IVC (Fig. 2). Thus, hatching ability of pig blastocyst was inversely corelated with the activation of Wnt/b-catenin signaling pathway. A previous study in mouse suggested that inhibition of Wnt/b-catenin during implantation might provide signal to blastocyst to begin hatching and to synchronize the preimplantation embryonic development with the differentiation of the uterus [27]. Next, we evaluated if the altered hatching ability of Dkk1- and LiCl-treated blastocysts were associated with altered development and differentiation of TE cells because of inhibition or activation of the Wnt/b-catenin signaling pathway. Because earliest epithelial differentiation of pig embryos are observed on Day 5 of IVC [36], we treated them with Dkk1 or LiCl for 24 h and then transferred to fresh media for further culture until Day 7. Results showed that Dkk1-treated blastocysts not only had increased (P < 0.05) hatching ability but also contained increased (P < 0.05) number of blastomeres compared with nontreated controls (Fig. 3). Furthermore, Dkk1-treated blastocysts displayed significantly lower ICM:TE ratio than
To study the role of the Wnt/b-catenin signaling pathway during hatching and early epithelial differentiation of pig blastocysts, we first examined the expression and distribution of b-catenin and DVL3 in the blastocyst during expansion and hatching. The b-catenin is a central component in the Wnt/b-catenin signaling pathway whereas DVL is a key transducer of divergent Wnt pathways and releases b-catenin from degradation during Wnt/ b-catenin signaling [31–33]. Previous studies have shown that, in an unstimulated state, b-catenin and DVL3 localizes as punctate vesicular structures in the cytoplasm of cells but translocates to the nucleus upon stimulation by Wnt signals [31,32,37–39]. We observed that, similar to those reported for mouse embryos [27], both DVL3 and b-catenin were clearly expressed in the nuclei of expanded blastocysts but disappeared in hatched blastocysts (Fig. 1). Thus, the Wnt/b-catenin signaling pathway was active in expanded blastocyst but was inhibited in hatched blastocysts. We also observed that b-catenin was also localized at the cell-cell borders of both expanded and hatched blastocysts. In tissue culture cells, cadherin had a potential role in sequestering signaling b-catenin at the plasma membrane [27,40]. These data therefore, suggest the
Fig. 2. Activation or inhibition of the Wnt/b-catenin pathway influences the hatching ability of pig blastocysts. Day 6 blastocysts were cultured in the absence (control; closed box) or presence of specific inhibitor (Dickkopf-1; open box) or activator (lithium chloride; shaded box) of Wnt/b-catenin signaling pathway and were evaluated for the percentage of hatched blastocysts after 24 h (Day 7) or 4 h (Day 8) of in vitro culture. Bars with different letters (a, b, c) differ significantly (P < 0.05) among respective groups.
signaling pathway by Wnt signals [34,35]. Blastocysts were evaluated for their hatching ability on Day 7 and 8 of IVC. The concentrations of the inhibitor/activator, used in the study, were based our preliminary experiments (Supplementary Table 1). The ability of Dkk1 and LiCl to modulate the Wnt/b-catenin signaling pathway was confirmed by evaluating the expression of b-catenin in the Dkk1/LiCl-treated blastocysts by immunocytochemistry. 2.4.3. Experiment 3: Effect of activating or inhibiting Wnt/ b-catenin signaling pathway on early epithelial differentiation in pig blastocysts In this set of experiments, to investigate the role of Wnt/ b-catenin signaling pathway on early epithelial differentiation of pig blastocysts, Day 5 embryos (stage at which early epithelial differentiation begins in pig embryos [36]) were cultured in the IVC medium supplemented with and without Dkk1 (10 mg/mL) or LiCl (10 mM) for 24 h (Day 6) and were subsequently cultured in the absence of Dkk1 and LiCl for the next 24 h (Day 7). Blastocysts were evaluated for their hatching ability, total cell number per blastocyst, ICM, TE, and the expression of CDX2. 2.5. Statistical analyses Statistical analyses were carried out using SAS software Version 8.2 (Statistical Analysis System Inc., Cary, NC, USA). Embryo development data was analyzed by chi-square test or ANOVA and the means of cell number were compared by ANOVA followed by Bonferroni multiple pair wise comparison after testing for normality (Kolmogorov– Smirnov test with Lillie for correction). Arc-sine transformation was performed before analyzing the percentage data. Data are presented as mean SEM. Differences at P < 0.05 were considered significant.
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Fig. 3. Activation or inhibition of the Wnt/b-catenin pathway influences the early epithelial differentiation in pig embryos. Day 5 embryos were cultured in the absence (control; closed box) or presence of specific inhibitor (Dickkopf-1 [Dkk1]; open box) or activator (lithium chloride [LiCl]; shaded box) of Wnt/b-catenin signaling pathway for 24 h and were subsequently cultured for another 24 h in the activator/inhibitor-free medium. (A) Time course of blastocyst expansion and hatching. (B) Hatching ability and total cell number/blastocyst. Bars with different letters (a, b, c) differ significantly (P < 0.05) among the respective groups. (C) Inner cell mass (ICM; blue color) and trophectoderm (TE; pink color) cells in blastocysts. (D) Expression and localization of CDX2 protein in pig blastocysts, as revealed by immunocytochemistry using specific antibody against CDX2. Green color shows the localization of bound primary antibodies captured with FITCconjugated secondary antibody. Red color shows the propidium iodide-stained nuclei. Magnification 400.
those of nontreated controls (Table 1; Fig. 3). In other words, Dkk1-treated blastocysts had a significantly higher proportion of TE cells to ICM cells compared with that of other groups (Table 1). In sharp contrast, LiCl-treated blastocysts showed significantly reduced hatching ability, contained reduced number of blastomeres, and had the lower proportion of TE cells to ICM cells than those of other groups (Fig. 3; Table 1). Of note, the change in proportion of ICM and TE cells in Wnt-inhibited blastocysts were related to increase in total cell number and increase in TE cells and the total number of ICM cells remained similar. Conversely, the change in proportion of ICM and TE cells in Wntactivated blastocysts were related to reduced total cell number and reduced TE cells. Thus, inhibition of Wnt/bcatenin signaling pathway probably increased the propensity of blastomeres toward the fate of TE and thereby, increased the total number of cells in blastocyst.
These results are in line with previous reports that activation of the Wnt signaling pathway was sufficient to maintain the pluripotency and proliferation of ICM-derived embryonic stem cells [4,25,26]. Differentiation and proliferation of TE cells is regulated by the transcription factor Cdx2 whose expression in turn is regulated by the transcription factor Tead4 [41]. Recent studies show that Hippo signaling pathway can regulate the spatial expression of Tead4 to regulate the expression of Cdx2 and TE fates of the blastomeres [2]. The Hippo signaling pathway can also interact with the Wnt/b-catenin signaling via the regulation of DVL activity and nuclear accumulation of b-catenin [3]. We found that modulation of Wnt/b-catenin signaling and b-catenin nuclear accumulation, by LiCl or Dkk1, not only affected the TE proliferation but also altered the expression of CDX2. The nuclei of LiCl-treated blastocysts showed reduced and irregular
Table 1 Effect of Dkk1 and LiCl on ICM and TE. Groups
Control Dkk1 LiCl
Cells, N (mean SEM)
Cells, % (mean SEM)
ICM:TE ratio
ICM
TE
ICM
TE
14.7 1.6a 13.7 0.8a 13.4 0.8a
65.1 3.7b 86.1 9.1a 48.0 3.2c
18.0 1.0b 14.5 1.0c 22.9 1.0a
82.0 1.0b 85.1 1.0a 78.7 1.5c
Values with different superscripts within each column differ significantly (P < 0.05). Abbreviations: Dkk1, Dickkopf-1; ICM, inner cell mass; LiCl, lithium chloride; TE, trophectoderm.
0.22 0.1a 0.17 0.1b 0.29 0.1c
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expression of CDX2 compared with those of control and Dkk1-treated blastocysts (Fig. 3). These data therefore, reinforce the possible involvement of Wnt/b-catenin signaling in regulating TE fate during the development of pig blastocyst. Future study should determine if the Wnt/bcatenin signaling pathway directly interacted with the Hippo signaling pathway in embryos to regulate the expression of Cdx2 and hence, the TE fate of the blastomeres. It might also be noted that, in our study, we used in vitro-produced embryos, which are known to have lower development potential than their in vivo counterparts. It remains to be determined if the Wnt/b-catenin signaling pathway behaved similarly in the in vivo-produced embryos. In conclusion, our study suggests that Wnt/b-catenin signaling might possibly be involved in the hatching and TE differentiation of pig blastocyst, providing the participation of Wnt/b-catenin signaling in preimplantation development. The results of this study will not only have implications in modulating the ICM:TE ratio of in vitro produced animal embryos (such as cloned embryos) but also will be of use in infertility treatment via the promotion of hatching in difficult-to-hatch blastocysts. Further studies should unravel the possible cross talk of Wnt/b-catenin signaling to other signaling pathways in hatching and TE fate determination. Acknowledgments This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (#PJ0090142012)” and BioGreen 21 Program (#PJ0080962012), Rural Development Administration, Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. theriogenology.2012.08.018. References [1] Zernicka-Goetz M, Morris SA, Bruce AW. Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo. Nat Rev Genet 2009;10:467–77. [2] Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, et al. The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 2009;16:398–410. [3] Varelas X, Miller BW, Sopko R, Song S, Gregorieff A, Fellouse FA, et al. The Hippo pathway regulates Wnt/beta-catenin signaling. Dev Cell 2010;18:579–91. [4] Sokol SY. Maintaining embryonic stem cell pluripotency with Wnt signaling. Development 2011;138:4341–50. [5] Tanaka SS, Kojima Y, Yamaguchi YL, Nishinakamura R, Tam PP. Impact of WNT signaling on tissue lineage differentiation in the early mouse embryo. Dev Growth Differ 2011;53:843–56. [6] van Amerongen R, Nusse R. Towards an integrated view of Wnt signaling in development. Development 2009;136:3205–14. [7] Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR, Bradley A. Requirement for Wnt3 in vertebrate axis formation. Nat Genet 1999;22:361–5. [8] Petersen CP, Reddien PW. Wnt signaling and the polarity of the primary body axis. Cell 2009;139:1056–68.
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Supplementary Fig. 1. Expression of b-catenin in expanded blastocysts cultured in the absence (control; A) or presence of specific inhibitor (Dickkopf-1 [Dkk1]; B) or activator (lithium chloride [LiCl]; C) of the Wnt/b-catenin signaling pathway. Green color shows the localization of bound primary antibodies captured with FITC-conjugated secondary antibody. Red color shows the propidium iodide [PI]-stained nuclei. In the negative control group (D), blastocysts were stained with secondary antibody (primary antibody omitted) similar to other experimental groups. Magnification 600.
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K.T. Lim et al. / Theriogenology 79 (2013) 284–290
Supplementary Table 1 Dose-dependent effect of LiCl on the hatching ability of Day 6 pig blastocysts. Concentration (mM)
Blastocysts, N
Hatching ability (mean SEM)
Control 0.01 0.1 1.0 10
32 33 32 34 32
56.3 36.4 28.1 11.8 3.1
2.5 2.7 1.7 3.6 1.3
(18) (12) (9) (4) (1)
Day 6 blastocysts were cultured in the absence (control) or presence of LiCl and evaluated for the percentage of hatched blastocysts after 24 h (Day 7). The experiment was repeated four times. Values within parenthesis indicate the number of embryos. Hatching ability for all concentrations differed significantly (P < 0.05). Abbreviation: LiCl, lithium chloride.